This invention relates to cytokines and cell signaling, and more particularly, to bovine tumor necrosis factor (TNF) receptor-I.
Tumor necrosis factor-xcex1 (TNF-xcex1) is a pleiotropic cytokine, and is produced by activated macrophages/monocytes and lymphocytes. TNF-xcex1 is a potent mediator in inflammatory and immune responses, including the recruitment of leukocytes to injured tissues during bacterial and other microbial infections, and following stimulation with inflammatory substances. When present in excessive quantities, however, TNF-xcex1 is known to cause tissue injury, and has been implicated in the pathology associated with several inflammatory and autoimmune diseases.
The biological effects of TNF are mediated through two distinct receptors, TNF-RI and TNF-RII (in humans, p55 and p75, respectively), which differ in sequence and molecular mass. TNF-RI is reported to be present at low levels in most, if not all, human cell types, and expression of the gene in humans can be upregulated by infection, interferons, and modulators of second messengers, such as phorbol esters. The extracellular portions of both receptors also exist in soluble forms, which are derived by proteolytic cleavage from the cell surface. The soluble TNF receptors retain the ability to bind TNF in solution. Activated cells often shed their TNF receptors, which can render the target cell less sensitive to the biological effects of TNF. Soluble TNF receptors have been identified in urine and sera from healthy individuals, and have been shown to be elevated in some chronic diseases and following inoculation with agents that induce TNF-xcex1 release.
The present invention describes the cloning and characterization of nucleic acid encoding the bovine tumor necrosis factor receptor-I (TNF-RI). Additionally, a soluble bovine TNF-RI was produced and demonstrated to be a potent inhibitor of bovine tumor necrosis factor xcex1 (TNF-xcex1). The availability of nucleic acid sequences encoding the bovine TNF-RI, particularly those nucleic acid sequences encoding a soluble form, provides a therapeutic compound for preventing and treating inflammatory diseases in bovine animals.
In general, the invention features an isolated nucleic acid, such as (a) the nucleotide sequence of SEQ ID NO:1; (b) a fragment of the nucleotide sequence of (a) encoding a functional polypeptide fragment; (c) a nucleotide sequence that is at least 85% identical to (a) or (b); or (d) a nucleotide sequence complementary to (a), (b) or (c). For instance, the nucleotide sequence may be at least 90% identical, or at least 95% identical, to (a) or (b). The nucleic acid may encode a bovine tumor necrosis factor receptor-I (TNF-RI), which may bind tumor necrosis factor (TNF). The nucleic acid may encode, for example, a TNF-RI having the amino acid sequence of SEQ ID NO:2.
The invention also features a nucleic acid such as (a) the nucleotide sequence shown in SEQ ID NO:3; (b) a fragment of the nucleotide sequence of (a) encoding a functional polypeptide fragment; (c) a nucleotide sequence that is at least 85% identical to (a) or (b); or (d) a nucleotide sequence complementary to (a), (b) or (c). The nucleic acid may encode a soluble bovine TNF-RI, which may bind TNF. The nucleic acid may encode, for example, a soluble bovine TNF-RI having the amino acid sequence of SEQ ID NO:4.
Additionally, there are provided vectors that include one or more of the above-described nucleic acids. The vector(s) may include elements necessary for expression of the above-described nucleic acids operably linked to the nucleic acid(s). The invention further provides host cells containing such vectors.
The invention also features isolated bovine TNF-RI and soluble bovine TNF-RI polypeptides. Additionally, antibodies that have specific binding affinity for the bovine TNF-RI and soluble bovine TNF-RI polypeptides are provided. A feature of the invention is a soluble bovine TNF-RI, which may include a bovine TNF-RI extracellular domain or fragments thereof. The soluble bovine TNF-RI may further bind TNF.
Yet another feature of the invention is an isolated nucleic acid encoding a fusion protein. Within the context of the present invention, the fusion protein may be encoded by a nucleic acid encoding a soluble bovine TNF-RI and a second nucleic acid sequence. For example, the second nucleic acid sequence may be an antibody or fragment thereof.
The invention also features a method of inhibiting TNF cytotoxicity in a bovine animal, and includes administering an effective amount of a polypeptide or a nucleic acid encoding the polypeptide to the animal. In the context of the present invention, the polypeptide is one or more soluble, functional polypeptide fragments of bovine TNF-RI that bind TNF and thereby inhibit TNF cytotoxicity in the bovine animal. For example, soluble, functional polypeptide fragments of bovine TNF-RI may be administered by direct infusion, and further, may be directly infused into the animal""s mammary gland. In the context of the present invention, inhibition of TNF cytotoxicity in the animal is useful, for example, to treat mastitis. The effective amount may be, for example, from about 1 xcexcg/kg body weight to about 1 mg/kg body weight.
Another feature of the invention is a pharmaceutical composition, including one or more soluble, functional polypeptide fragments of bovine TNF-RI and a pharmaceutically acceptable carrier. Also provided is a kit, which includes at least one unit dose of the above-described pharmaceutical composition.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable, but not necessarily preferred, methods and materials are described below. In addition, the materials, methods and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims.